Volume 21, Issue 2, Pages 338-347 (February 2013) Multifaceted Therapeutic Targeting of Ovarian Peritoneal Carcinomatosis Through Virus- induced Immunomodulation  Shashi Gujar, Rebecca Dielschneider, Derek Clements, Erin Helson, Maya Shmulevitz, Paola Marcato, Da Pan, Lu-zhe Pan, Dae-Gyun Ahn, Abdulaziz Alawadhi, Patrick WK Lee  Molecular Therapy  Volume 21, Issue 2, Pages 338-347 (February 2013) DOI: 10.1038/mt.2012.228 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Virus-mediated oncolysis and enhanced inherent antigen presentation molecule expression of OC cells. (a) ID8 cells infected in vitro with 1, 10 or 100 MOI of reovirus were harvested at 24, 48, and 72 hours, stained with annexin-V/7-AAD and then analyzed in flow cytometry for the detection of apoptotic cells. (b) Data shows representative profiles of ID8 cells at respective timepoints. (c) In addition, cells were stained with anti-MHC I (surface staining) and antireovirus antibodies (intracellular staining) at respective timepoints shown. (d) In addition, at 24 hours post-infection, cells were also analyzed in quantitative real-time PCR to quantitate the gene expression of selected genes (as shown) using the Livak and Schmittgen's 2-ΔΔCT method.36 Gene expression in LRV-treated cells was normalized against GAPDH gene expression, and then compared against that observed in PBS-treated cells to calculate fold change. In both b and d, LRV-treated samples were compared against PBS-treated samples using Student's t-test at 95% CI; ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Cumulative data from a single experiment is representative of atleast five other independent experiments. CI, confidence interval; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; LRV, live reovirus; MHC, major histocompatibility complex; MOI, multiplicity of infection; OC, ovarian cancer; ns, not significant; PBS, phosphate-buffered saline. Molecular Therapy 2013 21, 338-347DOI: (10.1038/mt.2012.228) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Overcoming OC-induced dysfunction of DCs. (a) ID8 cells and BMDCs were co-cultured together for 24 or 72 hours before infection with 10 MOI of reovirus for 48 hours. Cells were stained with antibodies against CD11c and either MHC II, CD86, CD80, and CD40 and analyzed using flow cytometry. Bars show respective percentages normalized against, and asterisks show statistical analysis as compared with, BMDCs cultured without ID8 or reovirus. The data is representative of at least five independent experiments. (b) 2 × 105 BMDCs were co-cultured with 2 × 105 ID8-ova cells and added with LRV/UVRV for 24 hours. Next, co-cultures were washed, added with 1 × 105 B3Z cells per well, incubated for additional 18–24 hours and then added with 0.15 mmol/l of CPRG for additional 4 hours. The breakdown of GPRG was read at 570 nm as a measure of CD8+ T-cell response. Statistical analysis was obtained by comparing readings from each experimental condition against that observed in only the B3Z wells; ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. The data is cumulative from three independent experiments. BMDC, bone marrow-derived DCs; CPRG, chlorophenol red-β-D-galactopyranoside; DC, dendritic cell; MHC, major histocompatibility complex; MOI, multiplicity of infection; ns, not significant; LRV, live reovirus; TAA, tumor-associated antigen; UVRV, UV-inactivated reovirus. Molecular Therapy 2013 21, 338-347DOI: (10.1038/mt.2012.228) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Virotherapy to target established advanced OC. (a) Female C57BL/6 mice were implanted i.p. with ID8 cells, injected with a regimen of PBS/UVRV/LRV as shown and then monitored for the (b) development of PC, (c) amount of ascitic fluid, and (d) survival. (b) Representative example of OC-PC–bearing animals treated with LRV against PBS-treated or non-OC–bearing animal. (c) Graphs represent volume of ascitic fluid collected from PBS/UVRV-treated animals on the day they were killed or LRV-treated animals on the days indicated. Data was analyzed with Student's t-tests at 95% CI; ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Statistics shown immediately on top of the bars was obtained by comparing the respective data against PBS control, while analysis above the horizontal lines were obtained through comparison of LRV-treated animals with that of UVRV-treated ones. (d) Survival in respective experimental groups (injected as per a) was calculated with the Kaplan–Meier survival method. The data is representative of three independent experiments. CI, confidence interval; d.p.f.i, days post first injection; d.p.l.i., days post last injection; i.p., intraperitoneally; LRV, live reovirus; ns, not significant; OC, ovarian cancer; PBS, phosphate-buffered saline; PC, peritoneal carcinomatosis; UVRV, UV-inactivated reovirus. Molecular Therapy 2013 21, 338-347DOI: (10.1038/mt.2012.228) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Initiation of antitumor immunological events. C57BL/6 mice were injected as per protocol shown in Figure 3a, and then killed at the indicated timepoints to obtain respective tissues. These samples were processed, RNA was extracted, purified, and reverse transcribed using random hexamers. (a) Quantitative real-time PCR was conducted with the gene-specific primers for reovirus, (b) MHC class I, β2M, TAP-1, and TAP-2, (c) CD3, CD4, CD8, and IFN-γ, and (d) TNF-α, IL-4, IL-6, and IL-10 followed by analysis using the Livak and Schmittgen's 2-ΔΔCT method.36 Graphs illustrate the linear fold change of the indicated mRNA replicates normalized to GAPDH and PBS control. Statistical analysis was performed with two-tailed, Student's t-test as described for Figure 3; ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Data is representative of three independent experiments. d.p.f.i., days post first injection; d.p.l.i., days post last injection; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; ns, not significant; PBS, phosphate-buffered saline; TNF, tumor necrosis factor. Molecular Therapy 2013 21, 338-347DOI: (10.1038/mt.2012.228) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Modulation of Tregs and MDSCs during therapeutic targeting of OC-PC. Female C57BL/6 mice were injected with ID8 cells and a regimen of PBS/LRV as per protocol shown in Figure 3a. Animals were killed on the respective days as indicated to obtain single cell suspensions from spleen, MLN or ascetic fluid. These cells were directly stained with either (a) Gr1/CD11b or (b) CD4/CD25/FOXP3 and analyzed using flow cytometry. (c,d) Alternatively, ID8-ova cells were used to initiate OC-PC. These animals were treated with a regimen of PBS/LRV as per protocol shown in Figure 3a. Splenocytes obtained from these animals, on respective days as indicated, were stimulated with SIINFEKL peptide and (c) analyzed for the ability of CD3+,CD8+ cells to produce IFN-γ through intracellular staining or the (d) capacity of CD3+ cells to proliferate in CFSE assay. Statistical analysis was performed as explained for Figure 3d; ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (e) On 10 d.p.l.i., splenocytes were also stimulated with UVRV-pulsed BMDCs and assayed in CFSE-based flow cytometric assay to monitor for antireoviral T-cell proliferation. The representative histograms show profiles of antitumor and antireoviral T-cell response, with proliferative index (PI) as a measure of activation as calculated using ModFit LT computer algorithm. All the results are representative of atleast five independent experiments. BMDC, bone marrow-derived DCs; DC, dendritic cell; d.p.f.i., days post first injection; d.p.l.i., days post last injection; IFN, interferon; LRV, live reovirus; MDSC, myeloid derived suppressor cell; MLN, mesenteric lymph node; ns, not significant; OC, ovarian cancer; PBS, phosphate-buffered saline; PC, peritoneal carcinomatosis. Molecular Therapy 2013 21, 338-347DOI: (10.1038/mt.2012.228) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Use of reovirus-based therapy for the prevention of PC development. (a) Female C57BL/6 mice were implanted with 3 × 106 ID8 cells on day 0 and then injected with a regimen of PBS/UVRV/LRV injections as shown in the schematic. The animals were monitored for the development of (b) ascites and (c) survival. The graphs show the cumulative data on the average days post-tumor implantation when ascites was first detected in respective groups of animals. Statistical analysis was performed through intergroup comparison using Student's t-tests at 95% CI; ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. CI, confidence interval; i.p., intraperitoneally; LRV, live reovirus; ns, not significant; PBS, phosphate-buffered saline; PC, peritoneal carcinomatosis; UVRV, UV-inactivated reovirus. Molecular Therapy 2013 21, 338-347DOI: (10.1038/mt.2012.228) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions